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Cryptophycin F – A potential cyanobacterial drug for breast cancer
Biomedicine & Aging Pathology 4 (2014) 229–234
Available online at
ScienceDirect www.sciencedirect.com
Original article
Cryptophycin F – A potential cyanobacterial drug for breast cancer Muniraj Sangeetha , Muniraj Menakha , Subramaniyan Vijayakumar ∗ PG and Research Department of Botany and Microbiology, AVVM Sri Pushpam College, Poondi, Thanjavur, 613503 Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 22 November 2013 Accepted 3 January 2014 Available online 18 February 2014 Keywords: Breast cancer ER␣ Cyanobacterial drugs Cryptophycin F Nostoc Glide Hex Insilico docking
a b s t r a c t Cancer is a group of disease characterized by uncontrolled cell divisions leading to abnormal growth of the tissue. Worldwide, breast cancer is the second most common type of cancer after lung cancer. Estrogen and progesterone bind to the receptors and may work with growth factors to cause cancer cell growth and proliferation. Estrogen receptor alpha (ER␣) is essential for mammary gland development and also plays a central role in breast cancer development by mediating estrogen induced cell proliferation. Multidisciplinary scientific investigations are making best efforts to combat this disease, but the perfect cure is yet to be achieved. The side effects of the available drugs make the need for the necessity of new improved drugs. Cyanobacterial resource offers a great scope for discovery of new drugs for breast cancer. Cyanobacterial novel bioactive compounds with unique biological activities may be useful in finding the potential drugs with greater efficacy, specificity for the treatment of human diseases. Molecular docking is a key tool in structural molecular biology and computer-assisted drug design. Nowadays, molecular docking approaches are routinely used in modern drug design to understand drug–receptor interaction. Computational techniques can strongly support and help the design of novel, more potent inhibitors by revealing the mechanism of drug-receptor interaction. Hence, the present study is interested to evaluate the interaction of the selected ligands with the breast cancer target receptor. From the study it is concluded that Cryptophycin F, a bioactive compound produced by the Nostoc is a promising potential drug for breast cancer. Crown Copyright © 2014 Published by Elsevier Masson SAS. All rights reserved.
1. Introduction Breast cancer is the second most common type of cancer, which starts in the cells of the breast in women and men [1]. Normal breast cells and most breast cancer cells have receptors that attach to circulating estrogen and progesterone. Estrogen and progesterone bind to the receptors and work with growth factors to cause cancer cell growth and proliferation [2]. Multidisciplinary scientific investigations are making best efforts to combat this disease, but the perfect cure is yet to be achieved. The side effects of the available drugs make the need for the necessity of new improved drugs [3]. ER␣ is essential for mammary gland development and plays a central role in breast cancer development by mediating estrogen induced cell proliferation. Estrogen receptors are a group of proteins found inside the cells, which are receptors that are activated by the hormone estrogen. Once activated by estrogen, the ER is able to translocate into the nucleus and bind to DNA to regulate the activity of different genes. It is a DNA-binding transcription factor [4]. There are two different forms of the estrogen receptor, usually referred to as ␣ and , each encoded by a separate gene, ESR1 and
∗ Corresponding author. Tel.: +09 44 3865923; fax: +04 37 4239438. E-mail address: svijaya [email protected] (S. Vijayakumar).
ESR2 respectively. Estrogen receptors are over-expressed in around 70% of breast cancer cases, referred to as ER-positive. Cancer treatments do not have potent medicine as the currently available drugs are causing side effects in some instances. Tamoxifen, a chemical drug, is taken orally as a tablet, which interferes with the activity of estrogen. Some of the most common side effects of tamoxifen are blood clots, strokes, uterine cancer, and cataracts. Raloxifene, another chemical drug, infrequently causes serious blood clots to form in the legs, lungs, or eyes. Other reactions experienced include leg swelling/pain, trouble breathing, chest pain, vision changes. The side effects of these drugs make the need for the necessity of new improved drugs [5]. Natural drug formulations for the prevention and treatment of cancer appeared in the last three decades, and the interest on natural sources of potential chemotherapeutic agents is continuing. Almost 60% of drugs approved for cancer treatment are of natural origin. Numerous types of bioactive compounds have been isolated from cyanobacteria. Several of them are currently in clinical trials or preclinical trials or undergoing further investigation. Although marine cynobacterial compounds are underrepresented in current pharmacopoeia, it is anticipated that the marine environment will become valuable source of novel compounds in the future, as it represents 95% of the biosphere [6]. However, development of marine floral compounds as therapeutic agents is still in its infancy.
2210-5220/$ – see front matter. Crown Copyright © 2014 Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biomag.2014.01.007
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M. Sangeetha et al. / Biomedicine & Aging Pathology 4 (2014) 229–234
Studies have clearly demonstrated that the cyanobacteria are an excellent source of novel drug discovery. Some marine organisms are proved to be the potent sources of drugs. Marine cyanobacteria are considered to be a group of potential organisms, which can be the richest sources of known and novel bioactive compounds including toxins with potential for pharmaceutical applications [7,8]. More than 50% of the marine cyanobacteria are potentially exploitable for extracting bioactive substances, which are effective in either killing the cancer cells by inducing apoptotic death. Cryptophycin is a potent cytotoxin produced by cyanobacteria of the genus Nostoc. It is also a promising drug in many cancer therapies. Thus, identification of new biologically active compounds is urgently required for development of new drugs. To fulfill the demand for new therapeutic drugs and to decrease the average costs involved in development, scientist should consider screening organisms from overlooked microbial source, cyanobacteria. Nowadays, molecular docking approaches are routinely used in modern drug design to understand drug-receptor interaction. It has been shown in the literature that these computational techniques can strongly support and help the design of novel, more potent inhibitors by revealing the mechanism of drug-receptor interaction [9]. Hence, the present study has planned to evaluate the interaction of the anticancer drugs administered for the target of breast cancer. 2. Material and methods The human estrogen receptor ER␣ structure (1R5K), responsible for breast cancer, was retrieved from Protein database (Pdb). The Pdb structures of commercially available drugs such as Raloxifene and Toremifene were retrieved from Chemspider database. The 2D structures of the analogue molecules of Arulide 1, Calothrixin B2, Caulerpenye, Cryptophycin F, Isomalgamide K, Malyngamide U, Malyngolide 1, Symplostatin 4 and Usneoidone 2 were retrieved from Chemspider database, which were later converted into 3-D structures using Swiss Pdb viewer. The analogue structures of the above said compounds were screened by using Schrodinger suite program to select a better ligand molecule against ER␣ receptor. The identified better compounds from Cyanobacteria and commercially available drug molecules were docked with the receptor molecule ER␣ using Hex software. The efficiency of the three ligands and their binding sites on the receptor were evaluated using Q-site finder. 3. Results ER␣, plays an important role in breast cancer development by mediating estrogen induced cell proliferation. At present commonly used antitumor drugs are Raloxifene and Toremifen in which side effects are so common. In breast cancer, ER␣ is to be controlled by effective antitumor compounds. Therefore, in the present investigation, suitable drug molecules with high binding affinity, which could be a possible lead molecule derived from cyanobacterial compounds are to be proposed.
Fig. 1. 3D structure of estrogen receptors alpha (ER␣).
as a good antigen and the molecule was more effective tumorogenic antigen. 3.2. Drug molecules Currently, Raloxifene and Toremifene are used as anticancer drugs for breast cancer. For cancer treatment, adequate potent medicines are not available. Marine cyanobacteria are considered to be the potential organisms as riche source of known and novel bioactive compounds, which are effective in either killing the cancer cells or affecting the cell signaling for cancer. Among the various members of marine Cyanobacteria, Calothrix, Lyngbya majuscule, Nostoc sp. and Symploca sp. are highly potential organisms having anticancer drug molecules and their analogues such as Arulide, Arulide B and C, Arulide 1-3; Calothrixin A, B, B2; Cryptopycin, 1, 5, 6, 16, 24, 38, 175, 176, 226, 326, 327 and 338, B, C, C1, D, E, F, G; Isomalgamide A, B and K; Malyngolide, 1, 2; Malyngamide A, C, H, I, L, M, N, O, O2, P, Q, R, S, T, T2, U, V, v2, W, 2-epi Malyngolide, Malyngolide dimmer and Symplostatin 1–4, Symplostatin analogue 1–3, 4, Symplostatin analogue 1 (Table 1; Figs. 4–12). From this analysis 6 Arulide, 3 calothrixin, 19 Cryptophycin, 3 Isomalgamide, 24 Malyngolide and 9 Symplostatin analogues were identified. When these analogues were docked with the receptor molecule ER␣ Cryptophycin-F showed a maximum Glide score indicating effective molecules against receptor tumor causing molecule (Table 1). From this result it is concluded that among the various cyanobacterial anticancer compounds Cryptophycin-F was identified as the best anti breast cancer molecule (Table 2). Currently, Raloxifene and Toremifene are used as anticancer drugs for breast cancer. In the present study Cryptophycin-F has been identified as anticancer drug for breast cancer, in addition to
3.1. Breast cancer receptor molecule ER␣ is a protein molecule containing three chains, A, B and C, each one formed by 261 amino acids (Figs. 1 and 2). In breast cancer, ER␣ is the functional protein and act as the antigen. In the present study, the antigenicity of the ER␣ molecule was analyzed using VaxiJen and TMHMM tool. The antigenicity of the receptor was 0.5039 which is greater than the threshold value of 0.5 for tumorogenic antigen and the antigen molecule was exomembrane in topology (Fig. 3). From the above facts, it was clear that the ER␣ is considered
Fig. 2. Ligand binding sites on ER␣.
M. Sangeetha et al. / Biomedicine & Aging Pathology 4 (2014) 229–234
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Fig. 3. Exomembrane topology of ER␣.
Fig. 4. Structure of Arulide 1.
Fig. 8. Structure of Malyngamide U.
Fig. 5. Structure of Calothrixin B2.
Fig. 9. Structure of Malyngolide1.
Fig. 6. Structure of Cryptopycin F.
the above two drugs. In order to find out which of the three drugs is more effective for the breast cancer treatment, molecular docking was carried out using Hex software. Docking results between human estrogen, ER␣ receptor and the conventional drugs (Raloxifene and Toremifene) and Cryptophycin-F were tabulated. These drugs have been used to target the human estrogen receptor. These drug molecules bound to the receptor and inhibit its function. The nature of the complex between the drug and the receptor complex was identified via docking and their relative stabilities for inhibition were evaluated using molecular dynamics and their binding affinities using free energy simulations. In this study,
Fig. 7. Structure of Isomalgamide K.
Cryptophycin-F showed a maximum e-value −319.77 and precision rate 55% (Fig. 13) against ER␣ molecule followed by Raloxifene having the e-value of −282.91. The third molecule Toremifene showed the least value of −248.61 (Table 3). When comparing the commercially available drugs currently used for the treatment, Cryptophycin-F, a compound of cyanobacterial origin is considered to be better and more effective in treating the ER␣ receptor causing breast cancer. The Cryptophycin-F molecule showed a high binding affinity with the major active site of receptor molecule ER␣ (Fig. 14).
Fig. 10. Structure of Symplostatin 4.
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Fig. 11. Structure of Raloxifene. Fig. 13. Docked structure of ER␣ with Cryptophycin F.
Table 3 Docking results of estrogen receptor ␣ with anticancer drugs. Antitumor drugs
Hex Docking score
Precision rate (%)
Cryptophycin F Raloxifene Toremifene
−319.11 −282.91 −248.61
55.1 37.8 0
High value highlighted in bold. Fig. 12. Structure of Toremifene.
Table 1 Docking results of estrogen receptor ␣ with analogues of cyanobacterial compounds using glide docking. Receptor name
Ligand name
Docking score
1R5k
Arulide 1 Calothrixin B2 Calothrixin B Calothrixin A Caulerpenye Caulerpenye 2 Cryptophycin F Cryptophycin G Isomalgamide K Malyngamide U Malyngamide L Malyngamide N Malyngamide S Malyngamide T Malyngamide C Malyngamide W Malyngamide P Malyngamide V Malyngamide T2 Malyngamide M Malyngamide V2 Malyngamide H Malyngamide 2 Malyngamide I Malyngamide O Malyngamide O2 Malyngolide 1 2-epi-malyngolide Malyngolide Symplostatin 4
−6.386 −9.842 −7.901 −7.843 −6.626 −6.566 −10.669 −9.242 −7.317 −8.328 −8.076 −7.784 −7.756 −7.602 −7.540 −7.468 −7.324 −7.308 −7.192 −7.155 −7.106 −6.986 −6.601 −5.458 −4.618 −4.259 −3.997 −3.532 −3.254 −9.329
Table 2 Docking results of estrogen receptor ␣ with cyanobacterial drugs. Cyanobacterial drugs
Source organism
Arulide1 Calothrixin B2 Cryptophycin F Isomalgamide K Malyngamide U Malyngolide1 Symplostatin 4
Lyngbya majuscule Calothrix Nostoc sp. Lyngbya majuscule Lyngbya majuscule Lyngbya majuscule Symploca sp.
High value highlighted in bold.
Glide docking score −6.386 −9.842 −10.669 −7.317 −8.328 −3.997 −9.329
Fig. 14. Binding of drug molecule (Cryptophycin F) at the major active site of ER␣ receptor molecule (Binding of drug molecule with the major active site of ERa shown inside the box).
4. Discussion Breast cancer is the most common cancer and one of the leading causes of death among women worldwide. It is a type of cancer originating from breast tissue by the stimulation of breast epithelial cells by the natural hormone, estrogen [10]. While the overwhelming majority of human cases occur in women, male breast cancer can also occur [1]. Breast cancer cells have receptors on their surface and in their cytoplasm and nucleus. Chemical messengers such as hormones bind to receptors, and this causes changes in the cell [11]. Estrogen receptors are a group of proteins found inside cells. They are receptors that are activated by the hormone estrogen [12]. Once activated by estrogen, the ER is able to translocate into the nucleus and bind to DNA to regulate the activity of different genes [4]. Estrogen receptors are over-expressed in around 70% of breast cancer cases, referred to as ER-positive. Estrogen and progesterone bind to the receptors and work with growth factors to cause cancer cell growth and proliferation [2]. Estrogen plays a crucial role in the breast cancer development which binds with estrogen receptor
M. Sangeetha et al. / Biomedicine & Aging Pathology 4 (2014) 229–234
and this complex triggers the DNA and gene activation leading to the proliferation of cells, there by leading to the breast cancer. The role of immune system is protecting the host against diseases by identifying and killing pathogens by the immune system. The basic criteria of a good antigen are that it must be exposed fully out side and must have antigenic score > 0.5 in the case of tumor [13]. Secreted and surface proteins of any given pathogen are mostly antigenic and are responsible for pathogenesis [14]. In the present study, ER␣ is an effective antigen by being exomembrane in topology and by having antigenic scores 0.5039, which is greater than the threshold value of 0.5 for tumorogenic antigen. Despite the pharmaceutical industry invested for high throughput screening systems, genomics and bioinformatic tools, the number of new entities reaching the market has declined from 53 to only 26 within a time span of 9 years (1996–2005). Currently, the pharmaceutical industry has to spend a lot of money to bring a new drug to market [15]. As a result, there is a scarcity of new therapeutic drugs reaching the market. Thus, identification of new biologically active compounds is urgently required for development of new drugs. To fulfill the demand for new therapeutic drugs and to decrease the average costs involved in development, scientists are screening organisms from overlooked microbial sources like Cyanobacteria [16]. One of the most important treatments currently available for cancer and other diseases is chemotherapy, which has limited effectiveness due to some serious life-threatening side effects and development of drug resistance cancer cells. In spite of the increasing success of chemotherapy, especially in achieving initial responses, it often fails in terms of long-term results because of the development of drug resistance by the cancer cells. The therapeutic efficacy and possible side effects vary among different agents. Some drugs may have excellent efficacy but its side effects are too serious. Also, they may have very limited supply and thus could be very expensive. However, new drug discovery is a long and expensive process. The side effects of the anticancer drugs not only prevent effective chemotherapy, but also compromise the quality of life of the patients [17]. One effective solution of the problem mentioned above can be using natural anticancer products for example cyanobacterial anticancer metabolites. The cryptophycins are among the most promising candidates for such a new drug. Cryptophycin 1, is one of the most potent natural anticancer drug from the geneus Nostoc sp. [18]. Ovarian carcinoma and breast carcinoma cells are less resistant to cryptophycin. This property may confer an advantage to cryptophycin in the chemotherapy of drug resistant tumors [19]. By using natural products, particularly cyanobacteria, as a source of chemical diversity, it is hypothesized that better inhibitors will be established and used to develop drugs for the treatment of cancer [20]. There is an urgent need of new anticancer drugs because tumor cells are developing resistance against currently available drugs, like vinca alkaloids and taxanes, which is thought to be a major cause of failure in the chemotherapeutic treatment of cancers. As a result, cancer is still among the leading cause of mortality worldwide [21]. Cryptophycins are potent anticancer agents produced by the cyanobacteria. Cryptophycin 1 was isolated from Nostoc sp. as anticancer agent, and thus it was found to be 100–1000 times more potent than currently available anticancer drugs. There are several analogues of cryptophycin either naturally isolated or chemically synthesized. Cryptophycin-52, a chemical analogue of cryptophycin-1, entered a clinical trial but produced only marginal activity. Currently two other analogues, cryptophicins 249 and 309, with improved stability and water solubility are being considered as second-generation clinical candidates. The enhanced efficacy of these compounds compared with that of Cr-52 has empowered
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new efforts to move these compounds into clinical trials [22]. In the present study Cryptophycin F was found to be most effective inhibitor over other ligand molecules such as, Arulide, Calothrixin, Isomalgamide, Malyngolide and Symplostatin against the breast cancer causing receptor ER␣ through molecular docking. 5. Docking Computational biology and bioinformatics have the potential not only of speeding up the drug discovery process thus reducing the costs, but also of changing the way drugs are designed. Rational Drug Design (RDD) helps to facilitate and speedup the drug designing process, which involves variety of methods to identify novel compounds. One such method is the docking of the drug molecule with the receptor (target). The site of drug action, which is ultimately responsible for the pharmaceutical effect, is a receptor (Computational Biology and Drug Discovery 2006). Docking is the process by which two molecules fit together in 3D space. Computer-aided drug design uses computational chemistry to discover, enhance, or study drugs and related biologically active molecules. The most fundamental goal is to predict whether a given molecule will bind to a target and if so how strongly. Molecular dynamics are most often used to predict the conformation of the small molecule and to model conformational changes in the biological target that may occur when the small molecule binds to it [23]. In this case, a large number of potential ligand molecules are screened. This method is usually referred as ligandbased drug design [24]. In the present investigation various drug molecules have been screened to find the fitting binding pocket of the receptor and effective potential ligand molecules were identified. Among the various cyanobacterial drugs such as, Arulide, Calothrixin, Isomalgamide, Malyngolide and Symplostatin against the breast cancer causing receptor ER␣ through molecular docking using Glide software. Out of three drug molecules, the cyanobacterial drug, Cryptophycin F two commercial drugs Toremifene and Raloxifene, Cryptophycin F was found to be more effective against the breast cancer receptor molecule, ER␣ using Hex software. 6. Conclusion From this study it is concluded that in breast cancer, ER␣ is the receptor molecule, which is considered as a good antigen. Currently, Raloxifene and Toremifene are used as anticancer drugs for breast cancer. Among the various members of marine cyanobacteria, Calothrix, L. majuscule, Nostoc sp. and Symploca sp. are highly potential organisms having anticancer drug molecules. Among the various cyanobacterial anticancer compounds, Cryptophycin F was identified as the best antibreast cancer molecule through molecular docking. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement The authors are thankful to the management of AVVM Sri Pushpam College (Autonomous), Poondi, for providing them necessary facilities and support to carry out this work. The authors are grateful to the UGC Major Research project, New Delhi, India (MRP R. No: 41-472/2012(SR)) for providing fund for this project. We specially express our thanks to the management of A.V.V.M. Sri Pushpam College (Autonomous), Poondi, for providing them necessary facilities and support to carry out this work.
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References [1] Sariego J. Breast cancer in the young patient. Am Surg 2010;76(12):1397–400. [2] Cosman. Selective estrogen-receptor modulator. Clin Geriatr Med 2003;19(2):371–9. [3] Alex Mathew J, Nixon Raj N.Docking studies on anticancer drugs for breast cancer using Hex. 2009. p. 18–20. [4] Levin ER. Integration of the extranuclear and nuclear actions of estrogen”. Mol Endocrinol 2005;19(8):1951–9, http://dx.doi.org/10.1210/me.2004-0390. PMC 1249516. PMID 15705661. [5] American Cancer Society. Cancer facts and figures; 2007. [6] Jimeno J, Faircloth G, Fernández Sousa-Faro JM, Scheuer P, Rinehart K. New marine derived anticancer therapeutics – a journey from the sea to clinical trials. Marine Drugs 2004;2:14–29. [7] Jha RK, Zi-Rong X. Biomedical compounds from marine organisms. Marine Drugs 2004;2:123–46. [8] Thajuddin N, Subramanian G. Cyanobacterial biodiversity and potential applications in biotechnology. Curr Sci 2005;89(1):47–57. [9] Xie L, Wang J, Bourne PE. In silico elucidation of the molecular mechanism defining the adverse effect of selective estrogen receptor modulators. PLoS Comput Biol 2007;3:e217. [10] Florescu A, Amir E, Bouganim N, Clemons M. Immune therapy for breast cancer in 2010 – hype or hope? Curr Oncol 2011;18(1):e9–18. PMC 3031364. PMID 21331271. [11] Sotiriou C, Pusztai L. Gene-expression signatures in breast cancer. N Engl J Med 2009;360:790–800. [12] Dahlman-Wright K, Cavailles V, Fuqua SA, Jordan VC, Katzenellenbogen JA, Korach KS, et al. International union of pharmacology. LXIV. Estrogen receptors. Pharmacol Rev 2006;58(4):773–81. PMID 17132854.
[13] Doytchinova IA, Flower DR. Bioinformatic approach for identifying parasite and fungal candidate subunit vaccines. Open Vaccine J 2008;1:22–6. [14] Plant LJ, Jonsson AB. Type IV pili of Neisseria gonorrhoeae influence the activation of human CD4+ T Cells. Infect Immun 2006;74(1):442–8. [15] Adams C, Brantner V. Estimating the cost of new drug development: is it really million dollars? Health Aff (Millwood) 2006;25:420–8. [16] Liu XJ, Chen F. Cell differentiation and colony alteration of Nostoc flagelliforme, an edible terrestrial cyanobacterium in different liquid suspension culture. Folia Microbiol 2003;48:619–25. [17] Feng SS, Chien S. Chemotherapeutic engineering: application and further development of chemical engineering principles for chemotherapy of cancer and other diseases. Chem Eng Sci 2003;58:4087–114. [18] Rohr J. Phytoestrogens derived from red clover: an alternative to estrogen replacement therapy? ACS Chem Biol 2006:47–750. [19] Smith CD, Zhang X, Mooberry SL, Patterson GML, Moore RE. A new microtubule depolymerizing agent from cyanobacteria. Cancer Res 1994;54: 3779–84. [20] Esmaeilzadeh M, Kazemzadeh F. Cancer treatment with using cyanobacteria and suitable drug delivery system. Ann Biol Res 2012;3(1):622–7. [21] Singh RK, Tiwari SP, Rai AK, Mohapatra TM. Cyanobacteria: an emerging source for drug discovery. J Antibiot 2011;64:401–12. [22] Yousong D, Seufert H, Zachary QB, David HS. Analysis of the cryptophycin P450 epoxidase reveals substrate tolerance and cooperativity. J Am Chem Soc 2008;130:5492–8. [23] Rajamani R, Good AC. Ranking poses in structure-based lead discovery and optimization: current trends in scoring function development’. Curr Opin Drug Discov Devel 2007;10(3):308–15. PMID 17554857. [24] Guner, Osman F. Pharmacophore perception, development, and use in drug design. La Jolla, Calif: International University Line; 2000, ISBN 0-9636817-6-1.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Cryptophycin F – A potential cyanobacterial drug for breast cancer Muniraj Sangeetha , Muniraj Menakha , Subramaniyan Vijayakumar ∗ PG and Research Department of Botany and Microbiology, AVVM Sri Pushpam College, Poondi, Thanjavur, 613503 Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 22 November 2013 Accepted 3 January 2014 Available online 18 February 2014 Keywords: Breast cancer ER␣ Cyanobacterial drugs Cryptophycin F Nostoc Glide Hex Insilico docking
a b s t r a c t Cancer is a group of disease characterized by uncontrolled cell divisions leading to abnormal growth of the tissue. Worldwide, breast cancer is the second most common type of cancer after lung cancer. Estrogen and progesterone bind to the receptors and may work with growth factors to cause cancer cell growth and proliferation. Estrogen receptor alpha (ER␣) is essential for mammary gland development and also plays a central role in breast cancer development by mediating estrogen induced cell proliferation. Multidisciplinary scientific investigations are making best efforts to combat this disease, but the perfect cure is yet to be achieved. The side effects of the available drugs make the need for the necessity of new improved drugs. Cyanobacterial resource offers a great scope for discovery of new drugs for breast cancer. Cyanobacterial novel bioactive compounds with unique biological activities may be useful in finding the potential drugs with greater efficacy, specificity for the treatment of human diseases. Molecular docking is a key tool in structural molecular biology and computer-assisted drug design. Nowadays, molecular docking approaches are routinely used in modern drug design to understand drug–receptor interaction. Computational techniques can strongly support and help the design of novel, more potent inhibitors by revealing the mechanism of drug-receptor interaction. Hence, the present study is interested to evaluate the interaction of the selected ligands with the breast cancer target receptor. From the study it is concluded that Cryptophycin F, a bioactive compound produced by the Nostoc is a promising potential drug for breast cancer. Crown Copyright © 2014 Published by Elsevier Masson SAS. All rights reserved.
1. Introduction Breast cancer is the second most common type of cancer, which starts in the cells of the breast in women and men [1]. Normal breast cells and most breast cancer cells have receptors that attach to circulating estrogen and progesterone. Estrogen and progesterone bind to the receptors and work with growth factors to cause cancer cell growth and proliferation [2]. Multidisciplinary scientific investigations are making best efforts to combat this disease, but the perfect cure is yet to be achieved. The side effects of the available drugs make the need for the necessity of new improved drugs [3]. ER␣ is essential for mammary gland development and plays a central role in breast cancer development by mediating estrogen induced cell proliferation. Estrogen receptors are a group of proteins found inside the cells, which are receptors that are activated by the hormone estrogen. Once activated by estrogen, the ER is able to translocate into the nucleus and bind to DNA to regulate the activity of different genes. It is a DNA-binding transcription factor [4]. There are two different forms of the estrogen receptor, usually referred to as ␣ and , each encoded by a separate gene, ESR1 and
∗ Corresponding author. Tel.: +09 44 3865923; fax: +04 37 4239438. E-mail address: svijaya [email protected] (S. Vijayakumar).
ESR2 respectively. Estrogen receptors are over-expressed in around 70% of breast cancer cases, referred to as ER-positive. Cancer treatments do not have potent medicine as the currently available drugs are causing side effects in some instances. Tamoxifen, a chemical drug, is taken orally as a tablet, which interferes with the activity of estrogen. Some of the most common side effects of tamoxifen are blood clots, strokes, uterine cancer, and cataracts. Raloxifene, another chemical drug, infrequently causes serious blood clots to form in the legs, lungs, or eyes. Other reactions experienced include leg swelling/pain, trouble breathing, chest pain, vision changes. The side effects of these drugs make the need for the necessity of new improved drugs [5]. Natural drug formulations for the prevention and treatment of cancer appeared in the last three decades, and the interest on natural sources of potential chemotherapeutic agents is continuing. Almost 60% of drugs approved for cancer treatment are of natural origin. Numerous types of bioactive compounds have been isolated from cyanobacteria. Several of them are currently in clinical trials or preclinical trials or undergoing further investigation. Although marine cynobacterial compounds are underrepresented in current pharmacopoeia, it is anticipated that the marine environment will become valuable source of novel compounds in the future, as it represents 95% of the biosphere [6]. However, development of marine floral compounds as therapeutic agents is still in its infancy.
2210-5220/$ – see front matter. Crown Copyright © 2014 Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biomag.2014.01.007
230
M. Sangeetha et al. / Biomedicine & Aging Pathology 4 (2014) 229–234
Studies have clearly demonstrated that the cyanobacteria are an excellent source of novel drug discovery. Some marine organisms are proved to be the potent sources of drugs. Marine cyanobacteria are considered to be a group of potential organisms, which can be the richest sources of known and novel bioactive compounds including toxins with potential for pharmaceutical applications [7,8]. More than 50% of the marine cyanobacteria are potentially exploitable for extracting bioactive substances, which are effective in either killing the cancer cells by inducing apoptotic death. Cryptophycin is a potent cytotoxin produced by cyanobacteria of the genus Nostoc. It is also a promising drug in many cancer therapies. Thus, identification of new biologically active compounds is urgently required for development of new drugs. To fulfill the demand for new therapeutic drugs and to decrease the average costs involved in development, scientist should consider screening organisms from overlooked microbial source, cyanobacteria. Nowadays, molecular docking approaches are routinely used in modern drug design to understand drug-receptor interaction. It has been shown in the literature that these computational techniques can strongly support and help the design of novel, more potent inhibitors by revealing the mechanism of drug-receptor interaction [9]. Hence, the present study has planned to evaluate the interaction of the anticancer drugs administered for the target of breast cancer. 2. Material and methods The human estrogen receptor ER␣ structure (1R5K), responsible for breast cancer, was retrieved from Protein database (Pdb). The Pdb structures of commercially available drugs such as Raloxifene and Toremifene were retrieved from Chemspider database. The 2D structures of the analogue molecules of Arulide 1, Calothrixin B2, Caulerpenye, Cryptophycin F, Isomalgamide K, Malyngamide U, Malyngolide 1, Symplostatin 4 and Usneoidone 2 were retrieved from Chemspider database, which were later converted into 3-D structures using Swiss Pdb viewer. The analogue structures of the above said compounds were screened by using Schrodinger suite program to select a better ligand molecule against ER␣ receptor. The identified better compounds from Cyanobacteria and commercially available drug molecules were docked with the receptor molecule ER␣ using Hex software. The efficiency of the three ligands and their binding sites on the receptor were evaluated using Q-site finder. 3. Results ER␣, plays an important role in breast cancer development by mediating estrogen induced cell proliferation. At present commonly used antitumor drugs are Raloxifene and Toremifen in which side effects are so common. In breast cancer, ER␣ is to be controlled by effective antitumor compounds. Therefore, in the present investigation, suitable drug molecules with high binding affinity, which could be a possible lead molecule derived from cyanobacterial compounds are to be proposed.
Fig. 1. 3D structure of estrogen receptors alpha (ER␣).
as a good antigen and the molecule was more effective tumorogenic antigen. 3.2. Drug molecules Currently, Raloxifene and Toremifene are used as anticancer drugs for breast cancer. For cancer treatment, adequate potent medicines are not available. Marine cyanobacteria are considered to be the potential organisms as riche source of known and novel bioactive compounds, which are effective in either killing the cancer cells or affecting the cell signaling for cancer. Among the various members of marine Cyanobacteria, Calothrix, Lyngbya majuscule, Nostoc sp. and Symploca sp. are highly potential organisms having anticancer drug molecules and their analogues such as Arulide, Arulide B and C, Arulide 1-3; Calothrixin A, B, B2; Cryptopycin, 1, 5, 6, 16, 24, 38, 175, 176, 226, 326, 327 and 338, B, C, C1, D, E, F, G; Isomalgamide A, B and K; Malyngolide, 1, 2; Malyngamide A, C, H, I, L, M, N, O, O2, P, Q, R, S, T, T2, U, V, v2, W, 2-epi Malyngolide, Malyngolide dimmer and Symplostatin 1–4, Symplostatin analogue 1–3, 4, Symplostatin analogue 1 (Table 1; Figs. 4–12). From this analysis 6 Arulide, 3 calothrixin, 19 Cryptophycin, 3 Isomalgamide, 24 Malyngolide and 9 Symplostatin analogues were identified. When these analogues were docked with the receptor molecule ER␣ Cryptophycin-F showed a maximum Glide score indicating effective molecules against receptor tumor causing molecule (Table 1). From this result it is concluded that among the various cyanobacterial anticancer compounds Cryptophycin-F was identified as the best anti breast cancer molecule (Table 2). Currently, Raloxifene and Toremifene are used as anticancer drugs for breast cancer. In the present study Cryptophycin-F has been identified as anticancer drug for breast cancer, in addition to
3.1. Breast cancer receptor molecule ER␣ is a protein molecule containing three chains, A, B and C, each one formed by 261 amino acids (Figs. 1 and 2). In breast cancer, ER␣ is the functional protein and act as the antigen. In the present study, the antigenicity of the ER␣ molecule was analyzed using VaxiJen and TMHMM tool. The antigenicity of the receptor was 0.5039 which is greater than the threshold value of 0.5 for tumorogenic antigen and the antigen molecule was exomembrane in topology (Fig. 3). From the above facts, it was clear that the ER␣ is considered
Fig. 2. Ligand binding sites on ER␣.
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Fig. 3. Exomembrane topology of ER␣.
Fig. 4. Structure of Arulide 1.
Fig. 8. Structure of Malyngamide U.
Fig. 5. Structure of Calothrixin B2.
Fig. 9. Structure of Malyngolide1.
Fig. 6. Structure of Cryptopycin F.
the above two drugs. In order to find out which of the three drugs is more effective for the breast cancer treatment, molecular docking was carried out using Hex software. Docking results between human estrogen, ER␣ receptor and the conventional drugs (Raloxifene and Toremifene) and Cryptophycin-F were tabulated. These drugs have been used to target the human estrogen receptor. These drug molecules bound to the receptor and inhibit its function. The nature of the complex between the drug and the receptor complex was identified via docking and their relative stabilities for inhibition were evaluated using molecular dynamics and their binding affinities using free energy simulations. In this study,
Fig. 7. Structure of Isomalgamide K.
Cryptophycin-F showed a maximum e-value −319.77 and precision rate 55% (Fig. 13) against ER␣ molecule followed by Raloxifene having the e-value of −282.91. The third molecule Toremifene showed the least value of −248.61 (Table 3). When comparing the commercially available drugs currently used for the treatment, Cryptophycin-F, a compound of cyanobacterial origin is considered to be better and more effective in treating the ER␣ receptor causing breast cancer. The Cryptophycin-F molecule showed a high binding affinity with the major active site of receptor molecule ER␣ (Fig. 14).
Fig. 10. Structure of Symplostatin 4.
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Fig. 11. Structure of Raloxifene. Fig. 13. Docked structure of ER␣ with Cryptophycin F.
Table 3 Docking results of estrogen receptor ␣ with anticancer drugs. Antitumor drugs
Hex Docking score
Precision rate (%)
Cryptophycin F Raloxifene Toremifene
−319.11 −282.91 −248.61
55.1 37.8 0
High value highlighted in bold. Fig. 12. Structure of Toremifene.
Table 1 Docking results of estrogen receptor ␣ with analogues of cyanobacterial compounds using glide docking. Receptor name
Ligand name
Docking score
1R5k
Arulide 1 Calothrixin B2 Calothrixin B Calothrixin A Caulerpenye Caulerpenye 2 Cryptophycin F Cryptophycin G Isomalgamide K Malyngamide U Malyngamide L Malyngamide N Malyngamide S Malyngamide T Malyngamide C Malyngamide W Malyngamide P Malyngamide V Malyngamide T2 Malyngamide M Malyngamide V2 Malyngamide H Malyngamide 2 Malyngamide I Malyngamide O Malyngamide O2 Malyngolide 1 2-epi-malyngolide Malyngolide Symplostatin 4
−6.386 −9.842 −7.901 −7.843 −6.626 −6.566 −10.669 −9.242 −7.317 −8.328 −8.076 −7.784 −7.756 −7.602 −7.540 −7.468 −7.324 −7.308 −7.192 −7.155 −7.106 −6.986 −6.601 −5.458 −4.618 −4.259 −3.997 −3.532 −3.254 −9.329
Table 2 Docking results of estrogen receptor ␣ with cyanobacterial drugs. Cyanobacterial drugs
Source organism
Arulide1 Calothrixin B2 Cryptophycin F Isomalgamide K Malyngamide U Malyngolide1 Symplostatin 4
Lyngbya majuscule Calothrix Nostoc sp. Lyngbya majuscule Lyngbya majuscule Lyngbya majuscule Symploca sp.
High value highlighted in bold.
Glide docking score −6.386 −9.842 −10.669 −7.317 −8.328 −3.997 −9.329
Fig. 14. Binding of drug molecule (Cryptophycin F) at the major active site of ER␣ receptor molecule (Binding of drug molecule with the major active site of ERa shown inside the box).
4. Discussion Breast cancer is the most common cancer and one of the leading causes of death among women worldwide. It is a type of cancer originating from breast tissue by the stimulation of breast epithelial cells by the natural hormone, estrogen [10]. While the overwhelming majority of human cases occur in women, male breast cancer can also occur [1]. Breast cancer cells have receptors on their surface and in their cytoplasm and nucleus. Chemical messengers such as hormones bind to receptors, and this causes changes in the cell [11]. Estrogen receptors are a group of proteins found inside cells. They are receptors that are activated by the hormone estrogen [12]. Once activated by estrogen, the ER is able to translocate into the nucleus and bind to DNA to regulate the activity of different genes [4]. Estrogen receptors are over-expressed in around 70% of breast cancer cases, referred to as ER-positive. Estrogen and progesterone bind to the receptors and work with growth factors to cause cancer cell growth and proliferation [2]. Estrogen plays a crucial role in the breast cancer development which binds with estrogen receptor
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and this complex triggers the DNA and gene activation leading to the proliferation of cells, there by leading to the breast cancer. The role of immune system is protecting the host against diseases by identifying and killing pathogens by the immune system. The basic criteria of a good antigen are that it must be exposed fully out side and must have antigenic score > 0.5 in the case of tumor [13]. Secreted and surface proteins of any given pathogen are mostly antigenic and are responsible for pathogenesis [14]. In the present study, ER␣ is an effective antigen by being exomembrane in topology and by having antigenic scores 0.5039, which is greater than the threshold value of 0.5 for tumorogenic antigen. Despite the pharmaceutical industry invested for high throughput screening systems, genomics and bioinformatic tools, the number of new entities reaching the market has declined from 53 to only 26 within a time span of 9 years (1996–2005). Currently, the pharmaceutical industry has to spend a lot of money to bring a new drug to market [15]. As a result, there is a scarcity of new therapeutic drugs reaching the market. Thus, identification of new biologically active compounds is urgently required for development of new drugs. To fulfill the demand for new therapeutic drugs and to decrease the average costs involved in development, scientists are screening organisms from overlooked microbial sources like Cyanobacteria [16]. One of the most important treatments currently available for cancer and other diseases is chemotherapy, which has limited effectiveness due to some serious life-threatening side effects and development of drug resistance cancer cells. In spite of the increasing success of chemotherapy, especially in achieving initial responses, it often fails in terms of long-term results because of the development of drug resistance by the cancer cells. The therapeutic efficacy and possible side effects vary among different agents. Some drugs may have excellent efficacy but its side effects are too serious. Also, they may have very limited supply and thus could be very expensive. However, new drug discovery is a long and expensive process. The side effects of the anticancer drugs not only prevent effective chemotherapy, but also compromise the quality of life of the patients [17]. One effective solution of the problem mentioned above can be using natural anticancer products for example cyanobacterial anticancer metabolites. The cryptophycins are among the most promising candidates for such a new drug. Cryptophycin 1, is one of the most potent natural anticancer drug from the geneus Nostoc sp. [18]. Ovarian carcinoma and breast carcinoma cells are less resistant to cryptophycin. This property may confer an advantage to cryptophycin in the chemotherapy of drug resistant tumors [19]. By using natural products, particularly cyanobacteria, as a source of chemical diversity, it is hypothesized that better inhibitors will be established and used to develop drugs for the treatment of cancer [20]. There is an urgent need of new anticancer drugs because tumor cells are developing resistance against currently available drugs, like vinca alkaloids and taxanes, which is thought to be a major cause of failure in the chemotherapeutic treatment of cancers. As a result, cancer is still among the leading cause of mortality worldwide [21]. Cryptophycins are potent anticancer agents produced by the cyanobacteria. Cryptophycin 1 was isolated from Nostoc sp. as anticancer agent, and thus it was found to be 100–1000 times more potent than currently available anticancer drugs. There are several analogues of cryptophycin either naturally isolated or chemically synthesized. Cryptophycin-52, a chemical analogue of cryptophycin-1, entered a clinical trial but produced only marginal activity. Currently two other analogues, cryptophicins 249 and 309, with improved stability and water solubility are being considered as second-generation clinical candidates. The enhanced efficacy of these compounds compared with that of Cr-52 has empowered
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new efforts to move these compounds into clinical trials [22]. In the present study Cryptophycin F was found to be most effective inhibitor over other ligand molecules such as, Arulide, Calothrixin, Isomalgamide, Malyngolide and Symplostatin against the breast cancer causing receptor ER␣ through molecular docking. 5. Docking Computational biology and bioinformatics have the potential not only of speeding up the drug discovery process thus reducing the costs, but also of changing the way drugs are designed. Rational Drug Design (RDD) helps to facilitate and speedup the drug designing process, which involves variety of methods to identify novel compounds. One such method is the docking of the drug molecule with the receptor (target). The site of drug action, which is ultimately responsible for the pharmaceutical effect, is a receptor (Computational Biology and Drug Discovery 2006). Docking is the process by which two molecules fit together in 3D space. Computer-aided drug design uses computational chemistry to discover, enhance, or study drugs and related biologically active molecules. The most fundamental goal is to predict whether a given molecule will bind to a target and if so how strongly. Molecular dynamics are most often used to predict the conformation of the small molecule and to model conformational changes in the biological target that may occur when the small molecule binds to it [23]. In this case, a large number of potential ligand molecules are screened. This method is usually referred as ligandbased drug design [24]. In the present investigation various drug molecules have been screened to find the fitting binding pocket of the receptor and effective potential ligand molecules were identified. Among the various cyanobacterial drugs such as, Arulide, Calothrixin, Isomalgamide, Malyngolide and Symplostatin against the breast cancer causing receptor ER␣ through molecular docking using Glide software. Out of three drug molecules, the cyanobacterial drug, Cryptophycin F two commercial drugs Toremifene and Raloxifene, Cryptophycin F was found to be more effective against the breast cancer receptor molecule, ER␣ using Hex software. 6. Conclusion From this study it is concluded that in breast cancer, ER␣ is the receptor molecule, which is considered as a good antigen. Currently, Raloxifene and Toremifene are used as anticancer drugs for breast cancer. Among the various members of marine cyanobacteria, Calothrix, L. majuscule, Nostoc sp. and Symploca sp. are highly potential organisms having anticancer drug molecules. Among the various cyanobacterial anticancer compounds, Cryptophycin F was identified as the best antibreast cancer molecule through molecular docking. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement The authors are thankful to the management of AVVM Sri Pushpam College (Autonomous), Poondi, for providing them necessary facilities and support to carry out this work. The authors are grateful to the UGC Major Research project, New Delhi, India (MRP R. No: 41-472/2012(SR)) for providing fund for this project. We specially express our thanks to the management of A.V.V.M. Sri Pushpam College (Autonomous), Poondi, for providing them necessary facilities and support to carry out this work.
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